The 70 kilodalton heat-shock-related proteins ("stress-70" proteins) comprise a pervasive family of molecular chaperones that are essential for the viability of living cells. Members of this protein family have been implicated in protein folding, transmembrane protein targeting, renaturation of denatured proteins, disassembly of specific supramolecular complexes such as clathrin, antigen presentation, and targeting of polypeptides for lysosomal proteolysis. The long-range goal of this work is to understand the structure and mechanism of stress-70 proteins in vitro.; this is a necessary prerequisite for understanding their in vivo biological activities. In the most simplistic view, these proteins are ATPases that bind and release "unstructured" polypeptides. They have a peptide binding activity, an ATPase activity, and a mechanism of coupling the ATPase cycle to peptide binding/release, presumably through a conformational change. Kinetics, mutagenesis and x-ray crystallography have been used to determine the ATPase mechanism. Small-angle x-ray scattering and fluorescence have been applied to the study of the magnitude and kinetics of the ATP-induced conformational change. Future efforts will focus on: 1. Mutants that should pre-empt the essential K+ ions in the nucleotide binding site will be constructed and characterized, to complete the studies of the ATPase mechanism. 2. The biochemical mechanism that couple the ATPase cycle with conformational change and peptide binding/release will be determined by measuring and correlating the kinetics of peptide binding and release with the kinetics of the conformational change and the individual steps of the ATPase reaction. 3. The amino acid preference of stress-70 proteins in peptide binding will be determined by measuring both equilibrium binding constants and kinetic "on" and "off" rates for tight-binding peptides, and then varying the type of amino acid at each site (-> Lys, Glu, Gly, Leu, Phe, Asn) and determining how different site-specific changes affect binding affinity. 4. Efforts will be made to crystallize full-length Hsc70 and its peptide binding domain.